Microbial degradation of mycotoxins

Alberts, Johanna Francina (2007-04)

Dissertation (PhD)--University of Stellenbosch, 2007.

Thesis

ENGLISH ABSTRACT: Aflatoxins are mycotoxins predominantly produced by the filamentous fungi Aspergillus flavus and Aspergillus parasiticus. Aflatoxin B1 (AFB1), the most abundant aflatoxin, is highly mutagenic, toxic, carcinogenic and teratogenic to humans and animals and is particularly correlated with the incidence of hepatocellular carcinoma in parts of Africa, China and South East Asia. In this regard aflatoxin is classified as a type I human carcinogen by the International Agency for Research on Cancer. Furthermore, aflatoxin contamination of food and feed is responsible for extensive economic losses due to loss of crops and farm animals. In spite of regulations regarding acceptable levels of aflatoxin in food, aflatoxin contamination remains a serious worldwide problem, especially in developing countries where it occurs predominantly in dietary staples. Inactivation of aflatoxin by physical and chemical methods has not yet proved to be effective and economic. However, biological detoxification offers an attractive alternative for eliminating toxins as well as safe-guarding the desired quality of food and feed. In this study, the biological degradation of AFB1 by bacteria and fungi was investigated. Several bacteria, including Rhodococcus spp., as well as white rot fungi have the potential to degrade a wide range of polycyclic hydrocarbon compounds due to the large repertoire of enzymes they produce and therefore the ability of some of these microorganisms to degrade AFB1 was investigated. Effective degradation of AFB1 by intracellular extracts of Mycobacterium fluoranthenivorans sp. nov. DSM 44556T, Nocardia corynebacterioides DSM 20151 and N. corynebacterioides DSM 12676 was demonstrated. Furthermore, AFB1 was effectively degraded by liquid cultures as well as intra- and extracellular extracts of Rhodococcus erythropolis DSM 14303. Significant (P<0.001) reduction in AFB1 was observed following treatment with R. erythropolis extracellular extracts with only 33.20% residual AFB1 after 72 h. Results indicated that the degradation by R. erythropolis DSM 14303 is enzymatic and that the enzymes are constitutively produced. The degradation of AFB1 when treated with R. erythropolis DSM 14303 extracellular extract coincided with a total loss of mutagenicity. In addition, treatment of AFB1 with culture fractions containing recombinant 2,3-dihydroxybiphenyl dioxygenase, which was produced through extracellular expression of the bphC1 gene of R. erythropolis DSM 14303 in Escherichia coli BL21, resulted in significant (P<0.0001) degradation (49.32%) and reduced mutagenic potency (42.47%) of the molecule. Significant (P<0.0001-0.05) degradation of AFB1 was obtained following treatment with culture extracts containing laccase enzyme produced by white rot fungi (17.10- 76.00%), purified fungal laccase from Trametes versicolor (1 U/ml, 87.34%) as well as with recombinant laccase produced by Aspergillus niger (118 U/L, 55.00%). Furthermore, treatment of AFB1 with purified fungal laccase enzyme (1 U/ml) resulted in loss of the mutagenic potency of the molecule. The decrease in the fluorescence and mutagenic properties of AFB1 following treatment with the microbial preparations imply changes to the furofuran- and/or lactone rings of the molecule. The current study contributes towards developing genetic engineered microbial strains which could be applied as an important bio-control measure. Such strains could exhibit multifunctional technological properties including degradation of AFB1, to significantly improve the quality, safety and acceptability of food.

AFRIKAANSE OPSOMMING: Aflatoksiene is mikotoksiene wat hoofsaaklik deur die filamentagtige fungi, Aspergillus flavus en Aspergillus parasiticus geproduseer word. Die algemeenste aflatoksien, aflatoksien B1 (AFB1), is hoogs mutagenies, toksies, karsinogenies en teratogenies vir mense en diere. Veral in sekere dele van Afrika, China en Suid-Oos Asië bestaan daar `n korrelasie tussen aflatoksien en die voorkoms van hepatosellulêre karsinoom en gevolglik word aflatoksiene as `n tipe I menslike karsinogeen deur die Internasionale Agentskap vir Kankernavorsing geklassifiseer. Aflatoksien kontaminasie in voedsel het ook `n ekonomiese impak as gevolg van verlies aan landbougewasse en diere. Ten spyte van maatreëls betreffende die toelaatbare vlakke van aflatoksiene in voedel, is aflatoksien kontaminasie steeds `n groot probleem wêreldwyd, veral in ontwikkelende lande waar dit hoofsaaklik in stapelvoedsel voorkom. Huidiglik is die inaktivering van aflatoksiene deur fisiese en chemiese metodes nie effektief en ekonomies nie. Daarteenoor bied biologiese tegnieke `n gunstige opsie vir die eliminering van die toksiene, terwyl die organoleptiese eienskappe van die voedsel steeds behoue bly. Hierdie studie fokus op die biologiese afbraak van AFB1 deur bakterieë en fungi. Verskeie bakterieë, insluitend Rhodococcus spp., sowel as witvrot fungi produseer `n verskeidenheid ensieme wat hulle in staat stel om `n wye reeks polisikliese hidrokoolstofverbindings af te breek en gevolglik is afbraak van AFB1 deur sommige van hierdie mikroörganismes bestudeer. Effektiewe afbraak van AFB1 deur intrasellulêre ekstrakte van Mycobacterium fluoranthenivorans sp. nov. DSM 44556T, Nocardia corynebacterioides DSM 20151 en N. corynebacterioides DSM 12676 is aangetoon. AFB1 is ook effektief in vloeibare kulture sowel as intra- en ekstrasellulêre ekstrakte van Rhodococcus erythropolis DSM 14303 afgebreek. `n Beduidende (P<0.001) afbraak van AFB1 is waargeneem na behandeling met R. erythropolis DSM 14303 ekstrasellulêre ekstrakte, met slegs 33.20% oorblywende AFB1 na 72 h. Resultate het getoon dat die afbraak deur R. erythropolis DSM 14303 ensimaties is en dat die ensieme konstitutief geproduseer word. Afbraak van AFB1 deur R. erythropolis DSM 14303 het ook tot `n totale verlies aan mutagenisiteit gelei. Verder het behandeling van AFB1 met rekombinante 2,3-dihidroksiebifenieldioksiginase fraksies wat geproduseer is deur ekstrasellulêre uitdrukking van die bphC1 geen van R. erythropolis DSM 14303 in Escherichia coli BL21, beduidende (P<0.0001) afbraak (49.32%) en vermindering in mutagenisiteit (42.47%) van die molekuul teweeggebring. Beduidende (P<0.0001-0.05) afbraak van AFB1 is verkry na behandeling met witvrot fungus kultuurekstrakte wat lakkase-ensiem bevat (17.10-76.00%), gesuiwerde lakkase geproduseer deur Trametes versicolor (1 U/ml, 87.34%), sowel as rekombinante lakkase geproduseer deur Aspergillus niger (118 U/L, 55.00%). Verder het die behandeling van AFB1 met gesuiwerde lakkase-ensiem (1 U/ml) gelei tot verlies aan mutagenisiteit van AFB1. Die afname in fluoressensie en mutageniese eienskappe van die AFB1-molekuul na behandeling met die onderskeie mikrobiese preparate dui op struktuurveranderings aan die furofuraan- en/of laktoonringe van die molekuul. Hierdie studie lewer `n bydrae tot die ontwikkeling van geneties gemanipuleerde mikrobiese rasse wat as `n belangrike biokontrole kan dien. Sulke rasse met multifunksionele tegnologiese eienskappe, insluitend die afbraak van AFB1, kan die kwaliteit, veiligheid en aanvaarbaarheid van voedsel verbeter.

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